Confocal microscope

Details Confocal microscope Confocal microscope

2000-03-21

2002-07-30

Nguyen, Thong (Department: 2872)

Optical: systems and elements

Compound lens system

Microscope

C359S234000, C359S368000, C359S385000

Reexamination Certificate

active

06426835

ABSTRACT:

CROSS-REFERENCE TO RELATED APPLICATIONS
This application is based upon and claims the benefit of priority from the prior Japanese Patent Applications No. 11-078281, filed Mar. 23, 1999; No. 11-080026, filed Mar. 24, 1999; No. 11-080028, filed Mar. 24, 1999; and No. 11-080202, filed Mar. 24, 1999, the entire contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
This invention relates to a confocal microscope adapted to observe and measure the micro-structure and the three-dimensional profile of a specimen by utilizing light.
Known typical confocal microscopes adapted to operate at high speed include those comprising a Nipkow's disk having a large number of pin holes arranged helically at intervals about ten times as large as their diameter. A confocal microscope comprising a Nipkow's disk is required to eliminate cross talk arising from adjacently located pin holes, and hence relatively large intervals have to be used in order to separate the pin holes from each other. The large intervals reduce the efficiency of utilizing the beam of light from the light source and, as a matter of fact, only 1% of the beam coming from the light source is utilized for the operation of the microscope. This means that the obtained image of the specimen is very dark.
R. Juskaitis, T. Wilson et al. proposed an improvement to confocal microscopes comprising a disk in “Efficient real-time confocal microscopy with white light sources”, Nature, Vol. 1, 383, October, 1996, pp. 804-806 and International Disclosure No. WO97/31282.
FIG. 1
of the accompanying drawings schematically illustrates a confocal microscope as proposed by T. Wilson et al.
Referring to
FIG. 1
, an optical lens
4
and a half mirror
6
are arranged on the optical path of the beam of light emitted from light source
2
, which may be a halogen light source or a mercury light source. A rotary disk
8
, an objective lens
10
and a specimen
12
are arranged on the optical path of the light beam reflected by the half mirror
6
.
Now, referring to
FIG. 2
, the rotary disk
8
has a random pin hole pattern section
8
a
where pin holes are randomly arranged and an aperture section
8
b
where light can pass freely. The random pin hole pattern section Ba and aperture section
8
b
are separated from each other by a pair of light blocking sections
8
c
,
8
d
that block any light trying to pass therethrough. The rotary disk
8
is linked to the shaft of a motor (not shown) by way of rotary shaft
14
so that it can be driven to rotate at a predetermined constant rate.
The beam of light reflected by the specimen
12
is made to enter CCD camera
18
by way of the objective lens
10
, the rotary disk
8
, the half mirror
6
and condenser lens
16
. The CCD camera
18
is controlled for the timing of its image pickup operation in synchronism with the rotary motion of the rotary disk
8
in such a way that it picks up two images of the specimen getting to it by way of the random pin hole pattern section
8
a and the aperture section
8
b
respectively.
The images output from the CCD camera
18
are stored in computer
20
. Of these, the image caught by the camera by way of the random pin hole pattern section
8
a
is a confocal image to which a non-confocal image (hereinafter referred to as composite image) is overlaid due to the fact that the density of pin holes is about ten times as high as that of pin holes of an ordinary Nipkow's disk.
Only a confocal image is obtained from subtraction of a composite image containing a confocal component and a conventional image obtained through the aperture section
8
b
. The calculated confocal image is displayed on the monitor
22
.
While only 0.5 to 1% of the beam coming from the light source is utilized in a Nipkow's disk type confocal microscope, 25 to 50% of the beam coming from the light source is utilized in a confocal microscope proposed by T. Wilson et al. so that they report that an image much clearer and brighter than an image obtained by a conventional Nipkow's disk type confocal microscope can be obtained by their camera.
Meanwhile, N. A. A. Neil, T. Wilson and R. Juskaitis, “A Light Efficient Optically Sectioning Microscope”, Journal of Microscopy, Vol. 189, pt. 2, (1998), pp. 114-117, describes an arrangement as shown in
FIG. 3
that is obtained by replacing the disk having randomly arranged pin holes of a known confocal microscope with a disk
24
having a linear pattern section
24
a
where a large number of light blocking areas and light transmitting areas (slits) are arranged linearly and alternately and an aperture section
24
b
where light can pass freely, the linear pattern section
24
a
and the aperture section
24
b
being separated by a pair of light blocking sections
24
c
,
24
d
adapted to block any light trying to pass therethrough. The authors claim that the proposed arrangement using such a disk can also provide a confocal image.
However, the above listed known disk scanning type confocal microscopes are accompanied by the following drawbacks.
While the disk scanning type confocal microscope proposed by T. Wilson et al. (International Disclosure No. WO 97/31282) provides an image tens of several times clearer and brighter than an image that can be obtained by a known Nipkow's disk type confocal microscope, the former is required to subtract the conventional image obtained by way of the light transmitting areas from the image (composite image) obtained by overlaying a non-confocal image to a confocal image getting to it by way of the pin hole section hole section.
However, the ratio of the brightness of the confocal image component to that of the non-confocal image component of a composite image varies as a function of the density of pin holes and the numerical aperture (NA) of the objective lens. On the other hand, the relationship between the brightness of the non-confocal image component of a composite image obtained by the random pin hole pattern section and that of the conventional image obtained at the aperture is not known. Therefore, it is difficult to obtain an optimal confocal image.
Additionally, a disk having linear slits as light transmitting areas does not provide any confocal image components in the direction parallel to the slits of the pattern but it does in the direction perpendicular to the slits of the pattern. In other words, the confocal effect of such a confocal microscope can vary depending on the direction of the image relative to the linear slits.
Still additionally, there may be cases where it is desirable to allow the non-confocal image to remain to a slight extent in addition to the obtained confocal image in order to vertically observe the specimen. However, with any known confocal microscopes adapted to obtain the confocal image by way of a subtracting process, the effect of the latter is automatically defined by the ratio of the area of the pin hole section and that of the light transmitting section (aperture section) so that the desk has to be replaced in order to change the effect of subtracting the conventional image from the composite image.
BRIEF SUMMARY OF THE INVENTION
In view of the above described problems of known confocal microscopes, it is therefore the first object of the present invention to provide a confocal microscope that can make the brightness of the non-confocal image component of the composite image and that of the conventional image substantially equal to each other in order to obtain an optimal confocal image.
The second object of the present invention is to provide a confocal microscope comprising a rotary disk having a light transmitting section (aperture section) formed by alternately arranging linear light blocking areas and light transmitting areas (slits), the rotary disk being adapted to obtain a relatively uniform confocal image.
The third object of the present invention is to provide a confocal microscope comprising a rotary disk and adapted to vary the ratio of the confocal image component to the non-confocal image component.
According to th

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